High phosphate directly affects endothelial function by downregulating annexin II.

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.

Firewall function of the endothelial glycocalyx in the regulation of sodium homeostasis.

Plasma sodium, slightly above normal and in presence of aldosterone, stiffens vascular endothelium and reduces nitric oxide release with the consequence of endothelial dysfunction. This process is mediated by epithelial sodium channels (ENaC) and, most likely, the endothelial Na(+)/K(+)-ATPase. Both, ENaC and Na(+)/K(+)-ATPase, are located in the plasma membrane of endothelial cells and embedded in the endothelial glycocalyx (eGC). This negatively charged biopolymer is directly exposed to the blood stream and selectively buffers sodium ions. We hypothesize that the glycocalyx could interfere with endothelial sodium transport when extracellular sodium varies in the physiological range. Therefore, we modeled the endothelial cell as a pump-leak system measuring changes of intracellular sodium in cultured human endothelial cells. Experiments were performed under low/high extracellular sodium conditions before and after enzymatic eGC removal, and with inhibition of Na(+)/K(+)-ATPase and ENaC, respectively. Three major observations were made: (1) eGC removal by heparinase treatment facilitates sodium to enter/exit the endothelial cells. (2) The direction of net sodium movement across the endothelial plasma membrane depends on the concentration of extracellular sodium which regulates both the Na(+)/K(+)-ATPase and ENaC activity. (3) Removal of eGC and inhibition of sodium transport modify the electrical resistance of endothelial cells. We conclude that the eGC serves as a potential "firewall" preventing uncontrolled access of sodium to the pump-leak system of the endothelial cell. After eGC removal, sodium access to the system is facilitated. Thus the pump-leak system could be regulated by ambient sodium and control vascular permeability in pathophysiological conditions.

Damage of the endothelial glycocalyx in chronic kidney disease.

BACKGROUND AND OBJECTIVES: The endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the luminal surface of endothelial cells, is considered as an intravascular compartment that protects the vessel wall against pathogenic insults in cardiovascular disease. We hypothesized that chronic kidney disease (CKD) is associated with reduced eGC integrity and subsequent endothelial dysfunction. METHODS & RESULTS: Shedding of two major components of the eGC, namely syndecan-1 (Syn-1) and hyaluronan (HA), was measured by ELISA in 95 patients with CKD (stages 3-5) and 31 apparently healthy controls. Plasma levels of Syn-1 and HA increased steadily across CKD stages (5- and 5.5-fold, respectively P < 0.001) and were independently associated with impaired renal function after multivariate adjustment. Furthermore, Syn-1 and HA correlated tightly with plasma markers of endothelial dysfunction such as soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular adhesion molecule-1 (sVCAM-1), von-Willebrand-Factor (vWF) and angiopoietin-2 (P < 0.001). Experimentally, excessive shedding of the eGC, evidenced by 11-fold increased Syn-1 plasma levels, was also observed in an established rat model of CKD, the 5/6-nephrectomized rats. Consistently, an atomic force microscopy-based approach evidenced a significant decrease in eGC thickness (360 ± 79 vs. 157 ± 29 nm, P = 0.001) and stiffness (0.33 ± 0.02 vs. 0.22 ± 0.01 pN/nm, P < 0.001) of aorta endothelial cell explants isolated from CKD rats. CONCLUSION: Our findings provide evidence for damage of the atheroprotective eGC as a consequence of CKD and potentially open a new avenue to pathophysiology and treatment of cardiovascular disease in renal patients.

Nanomechanics of the endothelial glycocalyx in experimental sepsis.

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.